Project Summary/Abstract: Long-distance signaling is a key feature of peripheral neurons. In the developing sympathetic nervous system, long-distance signals via the neurotrophin-receptor pair NGF-TrkA are transported in a signaling endosome (SE) from the distal axon back to the soma. Upon arrival in the soma, the SE signals for survival and, strikingly, traffics into the dendrite where it organizes post-synaptic densities. However, the trafficking mechanisms underlying SE transport into dendrites is unknown. There is thus a fundamental gap in the understanding of how trafficking and signaling of the SE are linked to create functional synaptic connections. The signaling cascades elicited by NGF-TrkA are well-understood. However, the trafficking events that it undergoes once it reaches the soma are severely understudied. The trafficking of the SE affects its signaling and vice versa. Therefore, uncovering how the SE is trafficked into dendrites using high resolution cell biological tools will move past the current barriers of mechanistically linking the physiology of signaling with the cell biology of endosome traffic and maturation. Our long-term goal is to understand how a retrograde signal is able to organize synapse formation. The rationale motivating this proposal is that retrogradely trafficked NGF-TrkA is able to transport into dendrites and initiate the clustering of post-synaptic densities. Additionally, inhibiting the MEK/MAPK pathway exclusively in the cell body prohibits SEs from entering dendrites, indicating a significant link between trafficking and signaling. Our lab recently showed that SEs undergo a novel trafficking event, signaling transcytosis, where the SE is externalized on the soma membrane and is subsequently re-internalized. The central concept of this proposal is that SEs regulate multiple downstream outcomes (survival vs PSD clustering) by undergoing distinct, regulated endosomal conversions in order to diversify their signaling capacity. Our preliminary data suggest the specific hypotheses that dendritic SEs are long-lived and non- degradative (Aims 1 and 2), and that the inhibition of the MEK/MAPK pathway prevents NGF-TrkA?s transition into this long-lived SE that can travel to dendrites (Aim 3). I will use innovative approaches including knockin mice, microfluidic devices, and quantitative single vesicle live imaging to address three specific aims: Aim 1) Determine the identity of dendritic SEs. What trafficking proteins (Rabs) associate with dendritic SEs? Aim 2) Determine how dendritic SEs are generated. What trafficking route do SEs take to reach the dendrites? Aim 3) Determine how interference with the MEK/MAPK pathway disrupts SE trafficking into dendrites. How does TrkA signaling affect its trafficking properties in the soma? The proposed research is significant because it will uncover the mechanisms of long-distance NGF signaling for dendrite development. The mechanisms uncovered in this work will have relevance as well to other long- distance signaling cascades via other growth factors in neurodevelopment.